The stage II transition of the mammary epithelium to copious milk secretion results from a precise cascade of events that transform the quiescent luminal epithelial cells of late pregnancy into active milk secreting cells. This transition is triggered by a fall in progesterone in the continued presence of high plasma prolactin and normal glucocorticoid. A critical event in the transition to lactation is the closure of tight junctions, erecting a physical barrier that provides functional separation of the lumen of the mammary gland from the interstitial space. Our goal is to determine the mechanistic linkage between progesterone withdrawal and tight junction closure. (1) We will determine changes in regulatory genes that accompany this transition using microarray analysis. (2) We will examine changes in tight junction proteins that occur during closure using proteomics (2D gel electrophoresis/mass spectrometry) to identify the relevant proteins.(3) The spatial and temporal distribution of candidate regulatory genes and proteins will be examined to determine whether they are expressed in a manner consistent with a role in regulation of tight junction closure. (4) Functional hypotheses about proteins that are expressed appropriately will be tested in vivo, utilizing intraductal injection of adenoviral vectors and transgenic and knock-out animals as appropriate. These experiments will provide insight into the mechanisms of tight junction regulation that should find broad biological applicability. In addition a better understanding of the mechanisms and regulation of tight junction closure during the early post-partum period will have significant clinical implications for the prevention and treatment of breast-feeding problems whose impact on child health is increasingly recognized.